The present invention is generally directed to cancer treatment and therapeutics, and more particularly to designing and synthesizing small interfering RNAs (siRNAs) having enhanced cytotoxicity.
Since their discovery over ten years ago, chemically-synthesized small interfering RNAs (siRNAs) have become the standard molecular biology tool for gene function studies. Their potential clinical application as therapeutic molecules is slowly becoming a reality due to improved delivery options. While significant challenges remain for the systemic delivery of siRNAs, several clinical trials have already documented the biological activity of siRNAs in target human tissues (References 1 and 2). The traditional approach has been to design a 19-mer double-stranded siRNA molecule consisting of two deoxythymidine (dT) nucleotide overhangs on either 3′-end (Reference 3). While dTdT overhangs have remained the standard overhang in siRNA synthesis, nearly any nucleotide can be used without incurring a deleterious effect on gene silencing (References 4 and 5). To enhance siRNA stability against nuclease degradation, nucleotides are often modified on the phosphate backbone and/or the ribose sugar moiety (Reference 6). These modifications are able to significantly extend the half-life of siRNAs in serum from minutes to days. In addition, these modifications are associated with a reduced number of off-target effects such as immune stimulation, passenger strand inactivation, and microRNA-like regulation (References 7 and 8). One issue that has yet to be addressed is the potential effect of these modified nucleotides on cellular metabolism following eventual intracellular degradation of the siRNA. Many anticancer and antiviral agents currently used in the clinical setting are nucleoside analogs (References 9 and 10). It is conceivable then that the modified nucleotides of siRNAs, once released from the siRNA molecule, might have potential impact on various cellular metabolic and signaling pathways.
Previous studies from our laboratory identified an siRNA molecule to potently and specifically inhibit thymidylate synthase (TS) expression (Reference 11). TS is a folate-dependent enzyme that catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by the reduced folate 5,10-methylenetetrahydrofolate to thymidylate (dTMP) and dihydrofolate (Reference 12). dTMP is then metabolized to dTTP, an essential precursor for DNA biosynthesis. Although dTMP can be formed by phosphorylation of thymidine via the thymidine kinase-catalyzed pathway, the TS-mediated formation of dTMP provides for its sole intracellular de novo synthesis. Given its central role in DNA biosynthesis and given the observation that TS inhibition results in suppression of cellular proliferation, TS represents an important target for cancer chemotherapy (References 13 and 14).
One of the hallmarks of a TS inhibitor compound, such as raltitrexed, pemetrexed, and 5-fluoro-2′-deoxyuridine (FdU), is the ability of exogenous thymidine to rescue against its cytotoxic and antitumor effects (References 15 and 16). We have previously demonstrated that the growth inhibitory effects of a specific TS-targeted siRNA was completely reversed by thymidine suggesting that the siRNA specifically targets TS with minimal off-target effects on other genes that might impact cell growth and proliferation (Reference 11). Recent studies from our laboratory have shown that the intracellular degradation of siRNA released dT nucleosides from the 3′-end overhang, which, in turn, rescued against the cytotoxicity resulting from TS inhibition (Reference 17). This dT release was able to reverse the growth inhibitory effects of TS siRNA as well as the cytotoxic effects of small molecule inhibitors of TS, such as raltitrexed and FdU.
Given the observation that the released nucleosides from siRNAs have biological effects, we hypothesized that siRNA molecules could be rationally designed to contain specific nucleosides that, once degraded intracellularly, would release cytotoxic analogs and thereby enhance the therapeutic potential of the siRNA. Herein, we demonstrate that the fluoropyrimidine nucleoside 5-fluoro-2′-deoxyuridine (FdU) can be directly incorporated into the siRNA backbone, leading to enhanced cytotoxic and apoptotic effects.